West Nile virus (WNV) was first detected in North America in 1999 during an outbreak of encephalitis in New York, and since that time has spread across the contiguous United States, Canada, Mexico, and the Caribbean. Recent work examining WNV evolution has identified a new genotype (WN02) that differs from the introduced genotype (NY99) by a single amino acid, a valine to alanine change at position 159 in the envelope (E) protein. Although the V159A change is not located within any of the known functional domains of the E protein, such as the predicted receptor binding domain or one of the domains required for efficient fusion, it is located three amino acids downstream from an N-linked glycosylation site present in the North American strains of WNV and may therefore affect its glycosylation. Previous studies have examined the effect of E protein glycosylation on viral infectivity and particle formation in vitro and on neuroinvasiveness in mice;however, to our knowledge there have been no studies examining the effect of glycosylation on the interactions between WNV and its mosquito vectors or avian reservoir hosts. This is important to examine because we previously found that WN02 viruses are transmitted by Culex mosquitoes two to four days earlier and cause significantly higher viremia titers and mortality in house sparrows than NY99 viruses. The experiments in this application will examine whether the presence or absence of the N-linked glycosylation site on the E protein of a prototype NY99 strain of WNV affects the virus-vector interactions. Using an infectious cDNA clone of WNV lacking the E protein glycosylation site, we will first examine whether glycosylation affects the ability of Culex mosquitoes to transmit infectious virus, viral replication within the mosquitoes, or viral binding to the mosquito midgut epithelium;we will then determine the effect of glycosylation on house sparrow viremia and mortality. We will also determine whether the consensus sequence differences between the two genotypes of WNV are sufficient to cause the observed phenotypic differences by introducing the V159A and noncoding mutations into the NY99 genotype cDNA clone of WNV, both individually and in combination, and examining whether these changes affect transmission of WNV by Culex mosquitoes or house sparrow viremia and mortality. Taken together, these studies will examine the effects of naturally occurring changes in viral sequence on the interactions of WNV with its natural vectors and reservoir hosts. It is interesting that the glycosylation site is not found in all WNV isolates throughout the world, but has been found in all North American isolates. The proposed studies will yield information on viral evolution and the adaptation of emerging viruses to naive environments, research critical to public health.